Conformational Ensembles from Experimental Data and Computer Simulations

Conformational Ensembles from Experimental Data and Computer Simulations

Poster Abstracts

56-POS Board 16 Conformational Dynamics of the Cop9 Signalosome-Cullin-2 RING E3 Ligase Supercomplex Probed by Hybrid Mass Spectrometry Andy M. Lau 1 , Sarah V. Faull 2 , Chloe Martens 1 , Zainab Ahdash 1 , Edward P. Morris 2 , Argyris Politis 1 . 2 Institute of Cancer Research, London, United Kingdom. 1 King's College London, London, United Kingdom, The Cullin-2 (CUL2) scaffold, along with RING-box binding protein 1 (RBX1) forms the core of a Cullin-2-RING Ligase (CRL2) complex. Further association with the VHL-EloB/C (VBC) adaptor complex, the CRL2VBC is responsible for cellular modulation of oxygen-dependent processes through regulation of the HIF-1α transcription factor. The activity of CRL2 VBC is regulated by the conjugation of the ubiquitin-like protein, NEDD8 (N8), through a process known as neddylation. Deactivation is achieved through complexing with the hetero-octameric Cop9 Signalosome (CSN) assembly which removes N8 from the CUL2 scaffold. Despite the importance of CSN-mediated CRL deactivation in regulating critical cellular processes, the precise and ubiquitous deactivation mechanism of CSN remains elusive to traditional structural biology techniques. Using a hybrid mass spectrometry (MS)-based strategy employing native, ion-mobility, chemical cross-linking and hydrogen deuterium exchange (HDX)-MS, combined with molecular dynamics simulations and integrative modelling with cryo-EM mapping, we have documented the highly dynamic conformational responses of CSN upon stimulation by both neddylated and deneddylated CRL2 VBC . Native MS has confirmed the formation of the CSN- CRL2 VBC supercomplex, while a mutation in the CSN5 subunit has allowed the generation of the CSN-CRL2 VBC ~N8 deneddylation intermediate. Integrative modelling of subunit connectivities via chemical cross-linking MS and electron density maps via cryo-EM have identified discrete architectures of CSN-CRL2 VBC and CSN-CRL2 VBC ~N8. Using differential HDX-MS and molecular dynamics simulations, we have further characterised the multiple layers of conformational dynamics imposed by binding of CRL2 VBC and CRL2 VBC ~N8. Our results show that neddylation of CRL2 VBC directly influences the mode of binding to CSN, while the deneddylation mechanism of CSN5 is indifferent to the neddylation status.

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